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After adding 340 L 4 C cold mixture of methanol and acetonitrile (ACN) (25:75, v/v) and 50 L of a 2 M D 9 -trimethylamine-N-oxide (Cambridge Isotopes Laboratories, Tewksbury, MA, USA) solution in methanol, the protein was precipitated by vortex-mixing for 30 s followed by 10 min incubation

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Vitamin C hydroxylates specific proline residues in hypoxia-inducible factor-I, which regulates hundreds of genes, and it also participates in the demethylation of DNA and histones and thereby influences the epigenome (26)

Taken together, the combination of these natural compounds with chemotherapeutic drugs like 5-FU not only helps alleviate chemotherapy-induced gut dysbiosis and toxicity, but may also enhance the overall therapeutic efficacy by improving the intestinal microenvironment and reinforcing immunomodulatory and anti-inflammatory mechanisms